Factor VIII, a plasma protein that participates in the blood coagulation cascade, is decreased or defective in individuals with hemophilia A. Factor VIII functions as a cofactor for the serine protease factor IXa in the surface-dependent conversion of zymogen factor X to the serine protease, factor Xa (Davie, E. W., Thromb. Haemost. 74:1-6 (1995); Lollar, P., Adv. Exp. Med. Biol. 386:3-17 (1995)). Deficiency of factor VIII activity results in a marked reduction of factor IXa activity and in the subsequent rates of factor Xa generated during the propagation phase of coagulation.
Factor VIII is synthesized as an ˜300-kDa single chain precursor protein (Wood et al., Nature 312:330-337 (1984); Toole et al., Nature 312:342-347 (1984)) with domain structure A1-A2-B-A3-C1-C2 (Vehar et al., Nature 312:337-342 (1984)). Factor VIII is processed to a series of divalent metal ion-linked heterodimers (Fass et al., Blood 59:594-600 (1982); Andersson et al., Proc. Natl. Acad. Sci. U.S.A. 83:2979-2983 (1986); Fay et al., Biochim. Biophys. Acta 871:268-278 (1986)) by cleavage at the B-A3 junction, generating a heavy chain (HC1) minimally represented by the A1-A2 domains; and a light chain (LC) consisting of the A3-C1-C2 domains. The A domains of factor VIII share homology with the A domains of factor V and the copper-binding protein, ceruloplasmin (Church et al., Proc. Natl. Acad. Sci. U.S.A. 81:6934-6937 (1984)). One mol of copper has been identified in factor VIII (Bihoreau et al., Eur. J. Biochem. 220:41-48 (1994); Tagliavacca et al., J. Biol. Chem. 272:27428-27434 (1997)).
People with deficiencies in factor VIII or antibodies against factor VIII who are not treated with factor VIII suffer uncontrolled internal bleeding that may cause a range of serious symptoms, from inflammatory reactions in joints to early death. Severe hemophiliacs, who number about 10,000 in the United States, can be treated with infusion of human factor VIII, which will restore the blood's normal clotting ability if administered with sufficient frequency and concentration. The classic definition of factor VIII, in fact, is that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A.
The development of antibodies (“inhibitors” or “inhibitory antibodies”) that inhibit the activity of factor VIII is a serious complication in the management of patients with hemophilia. Autoantibodies develop in approximately 20% of patients with hemophilia A in response to therapeutic infusions of factor VIII. In previously untreated patients with hemophilia A who develop inhibitors, the inhibitor usually develops within one year of treatment. Additionally, autoantibodies that inactivate factor VIII occasionally develop in individuals with previously normal factor VIII levels. If the inhibitor titer is low enough, patients can be managed by increasing the dose of factor VIII. However, often the inhibitor titer is so high that it cannot be overwhelmed by factor VIII. An alternative strategy is to bypass the need for factor VIII during normal hemostasis using factor IX complex preparations (for example, KONYNE®, Proplex®) or recombinant human factor VIIa. Additionally, since porcine factor VIII usually has substantially less reactivity with inhibitors than human factor VIII, a partially purified porcine factor VIII preparation (HYATE:C®) is used. Many patients who have developed inhibitory antibodies to human factor VIII have been successfully treated with porcine factor VIII and have tolerated such treatment for long periods of time. However, administration of porcine factor VIII is not a complete solution because inhibitors may develop to porcine factor VIII after one or more infusions.
Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These include a partially-purified factor VIII derived from the pooled blood of many donors that is heat- and detergent-treated for viruses but contain a significant level of antigenic proteins; a monoclonal antibody-purified factor VIII that has lower levels of antigenic impurities and viral contamination; and recombinant human factor VIII, clinical trials for which are underway. Unfortunately, human factor VIII is unstable at physiologic concentrations and pH, is present in blood at an extremely low concentration (0.2 μg/ml plasma), and has low specific clotting activity.
Hemophiliacs require daily replacement of factor VIII to prevent bleeding and the resulting deforming hemophilic arthropathy. However, supplies have been inadequate and problems in therapeutic use occur due to difficulty in isolation and purification, immunogenicity, and the necessity of removing the AIDS and hepatitis infectivity risk. The use of recombinant human factor VIII or partially-purified porcine factor VIII will not resolve all the problems.
The problems associated with the commonly used, commercially available, plasma-derived factor VIII have stimulated significant interest in the development of a better factor VIII product. There is a need for a more potent factor VIII molecule so that more units of clotting activity can be delivered per molecule; a factor VIII molecule that is stable at a selected pH and physiologic concentration; a factor VIII molecule that is less apt to cause production of inhibitory antibodies; and a factor VIII molecule that evades immune detection in patients who have already acquired antibodies to human factor VIII.
The present invention is directed to overcoming these and other deficiencies in the art.